This invention relates to the design and synthesis of antisense oligonucleotides which can be administered to inhibit the replication of cytomegalovirus and treat cytomegalovirus infections. These compounds can be used either prophylatically or therapeutically to reduce the severity of disease caused by cytomegaloviruses.
Cytomegalovirus (CMV) is infectious to humans of all ages beginning with gestation. CMV infection causes a wide spectrum of diseases including severe congenital malformations, a mononucleosis syndrome in adolescent and young adults, and fatal disseminated infection in immunosuppressed patients. Since CMV is so common as a latent virus in the general population and since cell-mediated immunity is the important element in host defense for controlling its proliferation, it is not surprising that CMV is a major pathogen in immune-compromised patients. While CMV is normally harmless in healthy individuals, CMV is a major cause of dysfunction in a wide variety of organs in AIDS patients, whose immune systems are no longer able to suppress it. Many AIDS patients have persistent CMV viremia. Some patients are viremic at a time when they are asymptomatic or have mild constitutional symptoms, but also have Kaposi's sarcoma. Almost all are viremic when they have had other life-threatening opportunistic infections.
Cytomegalovirus retinitis is a severe problem in immunocompromised patients and often leads to blindness. CMV retinitis may affect up to 40% of AIDS patients in the final stages of their disease, and treatment is difficult because the available drugs are toxic, costly and work best when given intravenously via an indwelling catheter. Cohen, J. Science 1995, 268, 368-9. Immunosuppressed patients are also very susceptible to CMV pneumonitis, which is one of the most lethal of human viral diseases, and gastrointestinal bleeding caused by CMV. Cohen et al. ibid. Cytomegalovirus may also play a role in the progression of HIV infection to AIDS by stimulating the transcription of the HIV long terminal repeats (LTR) in non-transformed, co-infected T cells. Histologic examination of adrenals and brains from AIDS patients has suggested that the adrenalitis, encephalitis and peripheral neuropathy observed were caused by CMV infection.
Effective therapy for CMV has not yet been developed despite studies on a number of antiviral agents. Interferon, transfer factor, adenine arabinoside (Ara-A), acycloguanosine (Acyclovir, ACV) and certain combinations of these drugs have been ineffective in controlling CMV infections. Based on reclinical and clinical data, foscarnet (PFA) and ganciclovir (DHPG) show limited potential as antiviral agents. DHPG studies have shown efficacy against CMV retinitis and colitis. DHPG seems to be well tolerated by most treated individuals, but the appearance of a reversible neutropenia, the emergence of resistant strains of CMV upon long-term administration, and the lack of efficacy against CMV pneumonitis limit the long term applications of this compound. The development of more effective and less-toxic therapeutic compounds and methods is needed for both acute and chronic use.
The present invention is directed to an alternative approach to the treatment of CMV infection, namely the inhibition of cytomegalovirus gene expression using antisense oligonucleotides. Antisense oligonucleotides targeted to CMV and therapeutic methods using these compounds have been disclosed in application Ser. No. 07/568,366 filed Aug. 16, 1990, now abandoned; Ser. No. PCT/US91/05815 filed Aug. 14, 1991; Ser. No. 07/927,506 filed Nov. 19, 1992, now issued as U.S. Pat. No. 5,591,720; Ser. No. 08/009,263 filed Jan. 25, 1993, now issued as U.S. Pat. No. 5,442,049, and Ser. No. 08/233,711 filed Apr. 26, 1994, now issued as U.S. Pat. No. 5,595,978, each of which is incorporated by reference herein in its entirety. A portion of this work has been published by Anderson et al. Antimicrob. Agents and Chemother. 1996, 40, 2004-2011. Antisense oligonucleotides directed to CMV are also disclosed in Pari et al., WIPO publication WO 95/32213 and Antimicrob. Agents Chemother. 1995, 39, 1157-1161.
An antisense phosphorothioate deoxyoligonucleotide drug, ISIS 2922 (SEQ ID NO: 22, Fomivirsen), has demonstrated clinical efficacy in halting progression of CMV retinitis in AIDS patients. Randomized, controlled, pivotal Phase III clinical trials of Fomivirsen are ongoing, for evaluation of Fomivirsen both as monotherapy and in combination with oral ganciclovir. Results have been presented from an ongoing, open-label clinical trial of Fomivirsen in patients with uncontrolled CMV retinitis. Twelfth International Roundtable on Nucleosides, Nucleotides and their Biological Applications, La Jolla, Calif., September 1996. Fomivirsen was administered by intravitreal injection, weekly for the first three weeks and every other week thereafter for maintenance. The drug produced rapid and prolonged disease remission when given at a 330 .mu.g dose in patients with advanced CMV retinitis who had failed other therapies. Prolonged disease remission was seen in patients who received Fomivirsen alone or as combination therapy with ganciclovir. The compound was found to be well tolerated, with a clinically acceptable safety profile characterized by manageable local ocular inflammatory complications and no systemic side effects.
In spite of this, it is desired to find an improved drug which can be administered less frequently, enhancing the patients' quality of life. It has now been surprisingly found that an analog of Fomivirsen which contains 2'-methoxyethoxy modifications is vastly more stable (has greatly increased residence time) in the retina compared to the deoxyphosphorothioate parent compound, Fomivirsen (ISIS 2922, SEQ ID NO: 22).